IMMUNOLOGY REVIEW ARTICLE

Innate immunity in vertebrates: an overview

Mario Riera Romo,1 Dayana Summary

P
erez-Mart
ınez2 and Camila
Castillo Ferrer3
1
Pharmacology Department, Centre of Marine
Bioproducts, Havana, 2Tumour Immunology
Direction, Centre of Molecular Immunology,
Havana, and 3Biochemistry Department,
Faculty of Biology, University of Havana,
Havana, Cuba
doi:10.1111/imm.12597
Received 3 December 2015; revised 10
February 2016; accepted 11 February 2016.
Correspondence: Mario Riera Romo, Centre
of Marine Bioproducts, Loma and 37 St,
Alturas del Vedado, PA 10600, Havana,
Cuba. Email: rieraromo@gmail.com
Senior author: Mario Riera Romo
Innate immunity is a semi-specific and widely distributed form of immu-
nity, which represents the first line of defence against pathogens. This
type of immunity is critical to maintain homeostasis and prevent microbe
invasion, eliminating a great variety of pathogens and contributing with
the activation of the adaptive immune response. The components of
innate immunity include physical and chemical barriers, humoral and
cell-mediated components, which are present in all jawed vertebrates. The
understanding of innate defence mechanisms in non-mammalian verte-
brates is the key to comprehend the general picture of vertebrate innate
immunity and its evolutionary history. This is also essential for the identi-
fication of new molecules with applications in immunopharmacology and
immunotherapy. In this review, we describe and discuss the main ele-
ments of vertebrate innate immunity, presenting core findings in this field
and identifying areas that need further investigation.
Keywords: defence mechanisms; evolutionary history; infection; innate
immunity; vertebrates.

innate mechanisms in non-mammalian species is essential

Introduction
Innate immunity is defined as the first line of defence
against pathogens, representing a crucial systemic
response to prevent infection and maintain homeosta-
sis.1–3 This type of immunity also has a critical role in
the activation and regulation of adaptive immunity.4
Despite its constitutive elements, innate defence mecha-
nisms have the capacity to develop an induced response
during primary infection and create inflammatory condi-
tions.5,6 This response is specific due to the cell surface
expression of pattern recognition receptors, which are
capable of recognizing complex polysaccharides,
glycolipids, lipoproteins, nucleotides and nucleic acids.4,7
The elements of innate immunity include external
physical barriers, humoral and cellular effector mecha-
nisms, components that are conserved among jawed ver-
tebrates (Table 1) with certain variations and specific
characteristics.2,3,8 Innate immunity has been extensively
studied in mammals in comparison to other vertebrates,
because of its significance for human health and veteri-
nary sciences.3,9–11 However, a major exploration of these
for comprehension of the complex events involved in
human innate immunity and is also crucial for the discov-
ery of new antimicrobial, antitumour and immunomodu-
latory molecules with therapeutic applications.
Recent studies have evaluated the innate immune
mechanisms in pathogen–host interactions and their phy-
logenetic conservation across animal species, revealing
new evidence that is changing the concept of innate
immunity. The former view of a non-inducible and pre-
existing innate immune response is now evolving to the
concept of a more specific immunity based on pattern
recognition receptor molecular recognition and even
memory development, leading to increased responses to
secondary infections.6,7,12–14 This innate immunological
memory depends mainly on natural killer (NK) cells and
macrophages, which provide protection against re-infec-
tion in a T/B-cell-independent manner.13 The develop-
ment of memory responses in innate cells, involves
epigenetic changes based on methylation and acetylation,
a functional reprogramming that induces cell reactivation
upon secondary stimulation.14 The memory in innate

Abbreviations: AMP, antimicrobial peptide; APP, acute phase protein; CRP, C-reactive protein; FV3, frog virus 3; IFN, inter-
feron; IL, interleukin; ITL, intraepithelial lymphocyte; NAb, natural antibody; NK, natural killer; TGF, transforming growth fac-
tor; TNF, tumour necrosis factor

ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139 125
M. Riera Romo et al.

Table 1. General overview of vertebrate innate immunity

Fish Amphibians Reptiles Birds Mammals References

Physical/chemical barriers
External protective structures + ++ ++ 15,16,18,19

External mucous secretions ++ ++ + + + 15,17,16,18

Specialized skin and mucosa  + + + ++ 2,16,20,24

Humoral innate immunity

Lysozyme + + + + + 20,21,33,34,35

Antimicrobial peptides + ++ + + ++ 15,20,22,37,40

Acute phase proteins ++ + + + ++ 9,10,47,49,51

Complement ++ + + + ++ 49,52,53,56,60

Cytokines + + + + ++ 65,66,68,70,71,73

Natural antibodies + + + + + 60,93,96,97,100

Cell-mediated innate immunity

Intraepithelial T lymphocytes + + + + + 11,25,26,27,28

Myeloid phagocytic cells + + + + + 73,75,104,108,112

Nuocytes + 85

Innate lymphoid cells + 113

Phagocytic B cells + + + ? 94,114,110

Non-specific cytotoxic cells + + ? + + 116,118,121,123

Mucosa-associated invariant T cells + 84

immunity has been described as a defence mechanism
present not only in vertebrate organisms, but also in
invertebrates such as plants and insects.12 In this context,
the study of innate immunity in non-mammalian verte-
brates could provide integrative evidence for the under-
standing of this changing type of immunity and its
evolutionary history.
In the current review, we examine the main defence
systems of vertebrate innate immunity, describing its
components, functions and properties. We also discuss
the evolutionary conservation and diversification of these
mechanisms, comparing them with their counterpart in
mammals and providing a background of what is known
and what needs further investigation in vertebrate innate
immunity.
Physical and chemical barriers
External structures as a protection strategy against patho-
gens are the primary mechanism of innate immunity,
which prevents microbe spreading and infection.1,2,15,16
Among different species these barriers have morphologi-
cal and anatomical variations, but they preserve the essen-
tial role of isolating the internal environment from
external factors, such as harmful substances and patho-
genic microorganisms2,15,17 (Fig. 1).
The skin is the main protective barrier in all living verte-
brates and consists of stratified epithelial layers with tight
junctions and structural proteins that provide mechanical
protection, preventing water loss and pathogen infiltration,
in addition to other sensory and excretory functions.16,18
Vertebrate integument contains two multilayered zones or
regions: the dermis, which is the most internal and the epi-
dermis, which is the most external and is in direct contact
with environmental factors and pathogens.16,18,19 This
basic morphology is shared by the five classes of verte-
brates, but the adaptation to their respective habitats has
led to the evolution of certain functional and structural
differences. For example, fish epidermis is protected by
ossified scales, actin-rich filaments called microridges and
localized keratin filaments, which are adequate mechanical
defences for aquatic environments and their associated
pathogens.15,16 The subsequent colonization of terrestrial
habitats by tetrapods led to the evolution of a more rein-
forced and specialized epidermis with an active keratiniza-
tion process and a resistant outer keratinized layer, the
stratum corneum, which is made of dead cells that are con-
tinually desquamated.16,18
In amphibians this corneous envelope contains a few
cell layers with a-keratin and does not form scales,
whereas in reptiles it is improved by a hard cornification
process that allows the formation of a thicker epidermis
with multiple layers of a- and b-keratin and rigid corneal
scales that confer additional protection.2,16,18,19 Avian
epidermis is similar to its reptilian predecessor and the
multiple a- and b-keratin layers, with a predominant b-
keratinized stratum corneum, is conserved.16,18,19 Birds
do not possess scales but instead they have feathers,
which are chemically and structurally resistant to patho-
gens and are also essential for aerial locomotion.18 Inter-
estingly, mammalian epidermis and its main appendices,
the hair, only contain a-keratin and keratin-associated
proteins, consequently they are mechanically less resistant
in comparison with scales or feathers.16,18,19 However,

126 ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139
 Innate immunity in vertebrates

Mucous secretion Antimicrobial peptides

Pathogen Lysozyme
External
environment Lysis

External protective
structures
Phagocytosis
Epithelial
barriers
Phagocytosis n
latio
ranu
Deg
Intraepithelial
T lymphocytes

Infected cell Lysis

Neutrophils
NK/NKT Macrophages /heterophils
cells Infiltration
Natural antibodies
Cell lysis ation
Neutraliz
Complement
Lysis proteins Capillary
vessel

Opsonization
Opsonization
Toxins

ocyte
Phagocytic B cells Leuc raction
o a tt
chem

Tissue

Toxin Acute phase proteins

inactivation

Figure 1. Integrative view of vertebrate innate immunity. Schematic illustration of the main innate defence systems in jawed vertebrates, which
represents its physical, humoral and cell-mediated components and the interactions between them, in a physiological context.

this loss of rigidity is compensated with a soft, flexible
and moistened stratum corneum, which is necessary
for mammalian locomotion and sensory functions. This
cornous layer has a specialized desquamation process and
a weakly acidic pH condition that prevent infections with
high efficiency.16,19
In addition, the skin of all vertebrates is constantly
lubricated with external secretions that maintain the epi-
dermal integrity and moisture and also exhibit important
antimicrobial properties.16,18 These secretions in aquatic
and semi-aquatic anamniotic vertebrates (fish and
amphibians) form a mucous cuticle that contains abun-
dant mucopolysaccharides and glycoproteins, whereas in
terrestrial amniotes (reptilians, birds and mammals) they
form a sebaceous fluid with glycolipids and lipids.15,17,18
Despite these differences in chemical composition, the
external secretions of all vertebrates are enriched in
microbicidal molecules, such as lysozyme and antimicro-
bial peptides (AMPs), which are an important innate
defence mechanism.2,3,15,20–22 The phylogenetic conserva-
tion and functional properties of these molecules are
discussed in a separate section.
The vertebrate skin also possesses specific cellular
responses to pathogen invasion. For instance, epithelial cells
can react to microbe contact by thickening and cellular
hyperplasia.8,18 Likewise, immature dendritic cells, called
Langerhans cells, infiltrate into the stratified layers of epi-
dermis where they extend dendritic prolongations, captur-
ing antigens and preventing local infection.2,16,20,23
Langerhans cells have been extensively characterized in
mammals but they are present in other vertebrates with
similar phenotypic and functional characteristics.16,23 Other
innate cells associated with these epithelial barriers are the
specific intraepithelial lymphocytes and macrophages that
destroy pathogens as well as transformed or infected
cells.1,2,23 The role of these innate immune cells is analysed
in detail in the Cell-mediated innate immunity section.
Mucosal membranes form another important epithelial
barrier conserved in vertebrate evolution that protects the
body cavities from invading pathogens.2,20,24 In fish, the
mucosal epithelium of the gills and the alimentary tract
protects the areas that are most vulnerable to microbe pen-
etration in aquatic environments.8 On the other hand,
more specialized mucosa of respiratory and gastrointestinal

ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139 127
M. Riera Romo et al.

tracts in terrestrial amniotes, prevent the infiltration of for-
eign particles and air-associated or ingested pathogens.16,20
In general, the mucosa of vertebrates exhibit basic bio-
chemical defences against microbes and parasites such as
the low pH, hydrolytic enzymes, the internal mucus layer
and the secretion of bioactive molecules that include lyso-
zyme, AMPs and pro-inflammatory cytokines.1,2,20,22,25
Other immune-related tissues, like the gut-associated lym-
phoid tissue, are also present in all jawed vertebrates and
contain high levels of infiltrating intraepithelial lympho-
cytes that destroy pathogens and preserve epithelial
integrity.3,11,26–29 In addition, the presence of symbiont
microbes, organized in communities called microbiomes,
have been reported in the majority of vertebrates; where
they are associated with the mucosal surfaces and the
skin.17,20 They inhibit the colonization by pathogenic
microorganisms, competing with them for attachment sites
and producing antimicrobial metabolites.17,20,24
Humoral innate immunity
Lysozyme
The mucous secretions and the plasma of all vertebrates
are enriched in proteins with antimicrobial activity
(Table 2). One of the most active is the lysozyme (mura-
midase or N-acetyl muramide glycanohydrolase), an
enzyme that lyses bacteria.1,3,21,30 This protein has been
found in fish,30 generally associated with the mucous
cuticle,8,15,31 whereas in amphibians like Rana pipiens
eight lysozyme isoforms were identified by chromato-
graphic and electrophoretic methods in the skin, serum,
liver, kidney, ovary and spleen, demonstrating lytic capac-
ity on a Micrococcus lysodeikticus-agar suspension.32 This
enzyme has also been identified in anurans like Xenopus
laevis33 and Xenopus tropicalis.31 In addition, different
lysozyme homologues have been isolated from reptiles
such as the Chinese soft-shelled turtle (Pelodiscus sinen-
sis), Asian soft-shelled turtle (Amyda cartilagenea) and
green sea turtle (Cheloni amydas), demonstrating lytic
ability against several strains of Gram-positive and Gram-
negative bacteria.34 In the same way, using an improved
lytic assay on Micrococcus lysodeikticus-agar plates, lyso-
zyme has been detected and quantified in the plasma of
captive birds, such as chickens (Gallus gallus), American
kestrels (Falco sparverius), cockatiels (Nymphicus hollandi-
cus) and zebra finches (Taeniopygia guttata) and free-liv-
ing birds, such as Clay-coloured thrushes (Turdus grayi),
blue-grey tanagers (Thraupis episcopus), crimson-backed
tanagers (Ramphocelus dimidiatus), variable seedeaters

Table 2. Detailed humoral immunity in jawed vertebrates

Molecule Function Organism1 References

20,22,37
a-Defensins Antimicrobial peptides B,M
20,22,37,38
b-Defensins Antimicrobial peptides R,B,M
22,37
h-Defensins Antimicrobial peptides M
15,20,37,39
Cathelicidins Antimicrobial peptides F, R, M
40
Hepcidin Antimicrobial peptide F, A, R, M
15,22,37,43,44
Cationic peptides Antimicrobial peptides F, A, R, M
9,10,48,49
C-reactive protein Acute phase protein F, A, M
10,48,49
Serum amyloid P Acute phase protein F, M
9,10,49,51
Serum amyloid A Acute phase protein F, R, B, M
10,46,52
Haptoglobin Acute phase protein F, B, M
9,10
a-1 acid glycoprotein Acute phase protein B, M
9,10,52
a-2 macroglobulin Acute phase protein F, M
9,10,49,51,52
Fibrinogen Acute phase protein F, R, B, M
23,49,51,52,53, 55
C3 Complement protein F, A, R, B, M
49,51,52,61
B factor C3 convertase F, A, B, M
49,51,52,53
C4 Complement protein F, B, M
23,49,51,52
C5 Complement protein F, A, B, M
52,53,55
C6 Complement proteins M
52,53,55
C1, C2, C7, C8, C9 Complement proteins F, M
65,66,67,70
Type I interferons Antiviral cytokines F, A, B, M
65,70,73,74
Type II interferons Phagocytosis stimulators F, A, R, B, M
71
Type III interferons Antiviral cytokines A, M
64,70,75,77,80
Interleukins Immunomodulatory cytokines F, A, R, B, M
49,64,70,75
Tumour necrosis factor-a Pro-inflammatory cytokine F, A, B, M
25,80,89,90
Transforming growth factor-b Immunomodulatory cytokine F, A, R, B, M
98,93,96,97,100
IgM Natural antibody F, A, R, B, M
1
F, fish; A, amphibians; R, reptiles; B, birds; M, mammals.

128 ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139

(Sporophila americana) and white-tipped doves (Leptotila
verreauxi).35 The bactericidal activity of these avian
plasma samples against Staphylococcus aureus and Escheri-
chia coli was evaluated, demonstrating lytic capacity
against both bacterial strains with higher levels for Escher-
ichia coli in all avian species.35 This enzyme is also
expressed in different mouse, rat and human tissues and
is secreted by glandular serous cells, surface epithelial
cells, and macrophages into the airway lumen.20,21 Mam-
malian lysozyme has been characterized at gene and pro-
tein levels in rodents, where different isoforms have been
identified.21,36 The bacteriolytic capacity of rat lysozyme
has been evaluated in vitro and in vivo, in transgenic
mice, demonstrating a major activity against Gram-posi-
tive bacteria like Pseudomonas aeruginosa and Staphylococ-
cus aureus.21 In humans, high concentrations of lysozyme
can be found in the airway surface liquid, where it con-
tributes to the microbicidal properties of nasal secre-
tions.20 This protein is also produced by neutrophils,
macrophages and specialized intestinal secretory cells
(Paneth cells), but can be secreted by gastrointestinal
epithelial cells during inflammatory and infectious condi-
tions, such as ulcerative colitis and Crohn’s disease.20
Recent phylogenetic analyses demonstrate the wide dis-
tribution of this enzyme across the whole animal king-
dom.33 The amino acid sequences of different lysozyme
types have been identified in vertebrates such as humans,
mice (Mus musculus), chickens (Gallus gallus), turtles
(Pelodiscus sinensis), frogs (Xenopus laevis), fish (Epine-
phelus coioides and Paralichthys olivaceus), in cephalochor-
dates like amphioxus (Branchiostoma japonicum) and in
several invertebrates and plants.33 The high conservation
of this protein in multicellular organisms is strong evi-
dence of its crucial role in innate immunity.
Antimicrobial peptides
The AMPs are another innate humoral defence that is
highly conserved in vertebrates. These molecules exhibit
antibacterial, antifungal and antiparasitic activity through
a wide variety of mechanisms including destabilization
and disruption of microbial membrane, pore formation,
protein aggregation, inhibition of intracellular targets,
interference in DNA transcription and blockade of pro-
tein synthesis and folding.22,37 The most conserved pep-
tides include defensins, cathelicidins and hepcidin.
Defensins and cathelicidins are present in the mucous
secretions of fish15 and reptiles like the European pond
turtle (Emys orbicularis)38 and the banded krait (Bun-
garus fasciatus).39 The b-defensin and cathelicidin-BF,
isolated from reptiles exhibit bactericidal capacity against
Salmonella typhimurium, Listeria monocytogenes, Escheri-
chia coli and methicillin-resistant Staphylococcus aureus
and antifungal activity against Candida albicans.38,39
Likewise, the defensin-like peptides and cathelicidins
ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139
Innate immunity in vertebrates
isolated from fish have potent antimicrobial activity
against different pathogens, such as bacteria, fungi,
viruses or parasites.15 The a- and b-defensins are also
found in birds and different mammals, such as cows,
goats, pigs, rats and mice.22,37 In humans, specific b-
defensins such as the human b-defensins 1, 2 and 3,
produced by epithelial cells and neutrophils, have been
identified.20,37 Other neutrophil-derived antimicrobial
peptides include azurocidins and a-defensins like the
human neutrophil peptides 1, 2 and 3.20 On the other
hand, in some mammals specific cathelicidins have been
found, such as PR-39 in pigs and LL-37 in humans20,37
and a unique type of defensin, the h-defensins, has been
isolated from the rhesus macaque (Macaca mulatta).22,37
The mammalian defensins and cathelicidins have been
shown to be an effective mechanism against bacterial
infections with Samonella typhimurium, group A strepto-
coccus, some Staphylococcus species and Pseudomonas
aeruginosa.20 Besides, they display multiple immunomod-
ulatory functions that include the stimulation of chemo-
kine production, inhibition of pro-inflammatory
cytokines, direct leucocyte chemotaxis and modulation
of host cell gene expression.20
Hepcidin, is possibly the most conserved AMP across
vertebrates. This small peptide enriched in cysteine resi-
dues is present in > 51 vertebrate species, where it acts
as an acute phase reactant synthesized by the liver (re-
viewed in ref. 40). The presence of hepcidin in birds is
debatable, but experiments based on mRNA expression
and searches in the EST and HTGS databases, did not
reveal any evidence that confirms the presence of this
gene in birds.40
The EST database (http://www.ncbi.nlm.nih.-gov/
nucest/) is a collection of short single-read transcript
sequences from GenBank. whereas the HTGS database
(http://www.ncbi.nlm.nih.gov/genbank/htgs) contains un-
finished DNA sequences generated by the high-throughput
sequencing centers using traditional clone-based sequen-
cing.
Other cationic peptides, with linear or helical structure,
have been found in different vertebrates. For example,
piscidin and pleurocidin have been isolated from fish,15,37
while pelovaterins are present in the eggs of the Chinese
soft-shelled turtle (Pelodiscus sinensis).41 Several AMPs
have been identified in amphibians, such as ascaphine-1
and ascaphine-8, isolated from Ascaphus truei and Asca-
phus montanus,42,43 bombinin H6, isolated from Bombina
bombina, Bombina orientalis and Bombina variegata44 and
alyteserin-1 and alyteserin-2, obtained from Alytes obstet-
ricans.45 Other examples are magainin 2, dermaseptin I,
brevinin IT, esculentin I, ranalexin and ranateurin I,
which were isolated from different amphibians.22,37 In
mammals, cationic peptides have also been found, such
as profenins in pigs and histatins in the saliva of humans
and some primates.37.
129
M. Riera Romo et al.
In the same way, AMPs derived from domains of other
proteins have also been described in vertebrates. Some
examples include hipposin, a histone-derived peptide
from Atlantic halibut (Hippoglossus hippoglossus L),30
lactoferricin from mammalian lactoferrin I, casodicin I
from human casein, buforin II, derived from the histone
2A, which is highly conserved among vertebrates, and
soluble antimicrobial domains of bovine lactoalbumin,
human hemoglobin, lysozyme and ovalbumin.22,37
Acute phase proteins
The vertebrate plasma and epithelial secretions contain
several proteins that are synthesized during infection and
inflammation, playing a crucial role in innate immunity
and host defence9,10,46 (Fig. 1). The main acute phase
proteins (APPs) described in mammals are C-reactive
protein (CRP), serum amyloid A and P, haptoglobin, a1-
acid glycoprotein, a2-macroglobulin, ceruloplasmin, fib-
rinogen and transferrin.10 Serum amyloid P and CRP are
pentraxins implicated in pathogen recognition, agglutina-
tion, opsonization and complement activation.10,47 CRP
is a major and moderate APP in many mammals, such as
sheep, cows, rabbits, dogs, mice, rats, humans and other
primates9,10 and different CRP homologues have been
identified in teleost fish,47,48 but not in reptiles or birds.
On the other hand, serum amyloid P seems to be a CRP
counterpart in some species.10,49 This lectin is a major
APP in mice, but has not been further identified in other
mammals.10 More recently, both CRP and serum amyloid
P were purified from the serum of the iridescent shark
(Pangasianodon hypophthalmus), demonstrating the
capacity to agglutinate rabbit erythrocytes and pathogenic
bacteria like Edwardsiella ictaluri and Aeromonas hydro-
phila.48 On the other hand, different pentraxins with CRP
domains have been described in amphibians of Xenopus
genus.47 Serum amyloid A is a more conserved APP that
has been identified in fish,49,50 reptiles,51 birds and mam-
mals,9,10 but not in amphibians. The expression of the
gene encoding the serum amyloid A protein is increased
in zebrafish (Danio rerio), rainbow trout (Onchorhyncus
mykiss) and Chinese soft-shelled turtle (Pelodiscus sinen-
sis) during bacterial infections.49–51 Serum amyloid A is a
major APP in mammals such as sheep, goats, cows,
horses, cats, dogs, pigs, mice and humans, but is a mod-
erate APP in chickens and non-human primates.10 This
protein is implicated in leucocyte chemoattraction and
regulation of the inflammatory response10 and is classified
as the major APP due to its high plasma levels during
inflammation.9,10 Interestingly, a trypsin inhibitor only
present in pigs is known as major acute phase protein
and is increased > 10-fold during infections.10
Haptoglobin and a1-acid glycoprotein are other impor-
tant acute phase reactants that are not present in all ver-
tebrates. Haptoglobin binds to free haemoglobin to
130
prevent oxidative damage and also displays antimicrobial
and immunomodulatory effects.10 This protein has only
been detected in fish and mammals,9,46,50,52 but recently
an avian counterpart of mammalian haptoglobin, called
PIT 54, was identified, which is induced in Escherichia
coli-infected chickens.46 Likewise, the a1-acid glycoprotein
has only been described in birds and mammals.9,10 Its
main function is the inactivation of harmful molecules
like lipopolysaccharide or different types of drugs.10
Other plasma proteins such as a2-macroglobulin,
ceruloplasmin, fibrinogen and transferrin, in addition to
their normal activity, can act as APPs and increase their
plasma levels during inflammation and infection.9,10 The
protease inhibitor a2-macroglobulin inactivates toxins
and proteolytic enzymes10 and has been identified in tel-
eost fish like Ictalurus punctatus52 and mammals such as
rats and primates,9,10 whereas ceruloplasmin is a copper-
binding protein that scavenges free radicals,10 and has
been identified as a positive APP in fish,52 birds46 and
mammals.10 Fibrinogen is involved in tissue repair and
is up-regulated during acute infections in fish,52 rep-
tiles,51 birds46 and mammals,9,10 while transferrin acts as
a negative APP in mammals,10 but is a positive acute
phase reactant in other vertebrates like fish52 and
birds.10 In contrast, albumin decreases its plasma levels
during acute infections or inflammatory conditions, rep-
resenting a negative APP among domestic animals like
chickens and mammals.10 The loss of albumin is caused
by renal and gastrointestinal changes during infection
and its hepatic synthesis decreases, representing a strat-
egy to preserve the unused amino acids for the produc-
tion of positive APPs.10
Complement system
The complement system is composed of serum proteins
that react against pathogens through a molecular cascade,
resulting in opsonization, enhanced inflammatory
response and formation of a membrane-penetrating
macromolecular pore, known as the membrane attack
complex, which produces microbial lysis1,3,53 (Fig. 1).
This system is better characterized in mammals, where
three activation pathways have been identified: the classi-
cal, the alternative and the lectin pathways.1,53–55 In the
classical pathway the proteolytic cascade is activated by
antigen–antibody immune complexes (generally with IgG
or IgM), whereas in the lectin pathway the recognition of
microbial surface carbohydrates by mannose-binding lec-
tin triggers the activation of mannose-binding-lectin-asso-
ciated serine proteases that initiate the complement
proteolytic cascade.53 The alternative pathway, on the
other hand, is self-activated by spontaneous hydrolysis of
the complement protein C3 at the microbial mem-
brane.1,53 Different studies in vitro on immobilized pro-
teins and in vivo in mouse models have revealed a novel
ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139

role of properdin in the activation of the alternative path-
way.53 Other evidence demonstrates the involvement of
coagulation-associated proteases like kallikrein and
thrombin in the complement proteolytic cascade, indicat-
ing a functional connection between these molecular
pathways.53 In immunodeficient patients and specific
knockout mice, the relevance of the complement system
for mammalian immunity has been evaluated,
demonstrating that several complement proteins or their
proteolytic fragments are capable of activating B- or T-
cell function and are also crucial for the innate immune
response against bacterial and viral pathogens, such as
Streptococcus pneumonia, Pseudomonas aeruginosa, Neisse-
ria meningitidis, Haemophilus influenza, herpes simplex
virus and West Nile virus (reviewed in ref. 53).The reac-
tivity and antimicrobial properties of the serum comple-
ment has also been evaluated in different vertebrates. For
instance, complement-mediated opsonization and anti-
body-mediated complement activation have been
described in teleost fish, such as common carp (Cyprinus
carpio), catfish (Ictalurus punctatus) and salmonids.8 Like-
wise, the serum haemolytic capacity has been studied in
different amphibians like Rana pipiens and Lithobates ber-
landieri.56 This study demonstrates that lytic activity
against heterologous cells is stimulated by antibodies,
reaches the levels reported for mammals and is influenced
by heat, EDTA, hydrazine, carrageenan and antigen–anti-
body precipitate to a similar extent as the complement of
some mammals, like pigs.56
The serum of reptiles also exhibits antimicrobial prop-
erties. Some comparative studies between the human
serum complement and the complement of the American
alligator (Alligator mississippiensis) demonstrate a major
range of action against Gram-negative bacteria for the
alligator serum.57 The complement of Alligator mississip-
piensis displays antiviral activity against human immun-
odeficiency virus type 1, West Nile virus and herpes
simplex virus,58 and additionally, amoebicidal properties
against various species of Naegleria and Acanthamoeba
that have been reported as resistant to human
complement lysis.59 In birds, evidence shows that the
complement system is also responsible for the lysis of
heterologous cells. Matson et al. in 200560 evaluated the
haemolytic capacity on rabbit erythrocyte suspension of
the sera from several bird species, including American
kestrel (Falco sparverius), Japanese quail (Coturnix cotur-
nix japonica), zebra finch (Taeniopygia guttata), mourning
dove (Zenaida macroura), waved albatross (Phoebastria
irrorata), grey catbird (Dumetella carolinensis) and com-
mon grackle (Quiscalus quiscula). This work demonstrates
that haemolysis decreases with heat, which indicates that
it is mediated by the complement system. A comparison
between subjects of different age and species also indi-
cates that complement-mediated lysis is affected by age
and varies among species.60
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Innate immunity in vertebrates
The mammalian complement system has been exten-
sively studied from a molecular perspective and nine
complement proteins have been identified: C1, C2, C3,
C4, C5, C6, C7, C8 and C9.1,53,55 Several complement
inhibitors detected in mammals impair the complement
cascade activation on host cell membranes and ensure the
specificity of this innate mechanism.53 The identified
mammalian inhibitors include Factor I, involved in C3b
and C4b proteolysis, the decay-accelerating factor, com-
plement receptor 1 (CD35), Factor H and C4-binding
protein, which are enzymatic inhibitors of C3 convertase
activity.53 Other inhibitors that impair the assembly of
the membrane attack complex, preventing C9 binding
and polymerization are CD59, vitronectin and S pro-
tein.53 In other vertebrates the presence of complement
proteins has been described, confirming that this mecha-
nism is preserved among vertebrates. Key components of
the complement system, such as C3 and C5, have been
isolated from some anurans of the genus Xenopus23 and
have been detected as down-regulated genes in teleosts
like Onchorhyncus mykiss during infection with Yersinia
ruckeri O1.49 In other osteichthyes like catfish (Ictalurus
punctatus), several complement proteins such as C1, C3,
C4, C5, C7, C8 and C9 have been identified.52 In addi-
tion, the B factor that is a crucial element of the C3 con-
vertase cascade implicated in alternative C3 activation has
been identified in Onchorhyncus mykiss,49 Ictalurus punc-
tatus,52 Silurana (Xenopus) tropicalis and Xenopus laevis.61
Similarly, different proteins of this enzymatic cascade
like C3, factor B, C4 and C5, have been identified in
domestic birds like chickens.62 In contrast, the molecular
composition of the complement cascade of reptiles
remains elusive and has only been identified the
complement protein C3.51
Cytokines
Cytokines are proteins that mediate the communication
between cells and are essential for the immune response.63
These soluble factors are one of the most conserved ele-
ments of vertebrate innate immunity. Cytokines are subdi-
vided into different classes according to their genomic
encoding regions, target receptors, associated signalling
pathways and biological functions; these classes include
interferons (IFNs), interleukins (ILs), tumour necrosis fac-
tors (TNFs) and transforming growth factors (TGFs).1,63,64
Although the cytokines characterized to date have been
cloned and extensively studied in mice, many of them are
present in other vertebrates with similar functions.
Interferons are mainly involved in antiviral defence,
although they display additional immunomodulatory
function.63–65 In mammals, three types of IFNs have been
identified so far: type I IFNs, which include IFN-a, IFN-
b, IFN-x, IFN-j and IFN-e (reported in humans), type II
IFNs (IFN-c) and type III IFNs (IFN-k).65 Type I IFNs
131
M. Riera Romo et al.
represent a conserved antiviral system in vertebrates but
they have a different genomic organization in lower and
higher vertebrates.66 Fish and amphibian type I IFNs are
encoded by characteristic five-exon/four-intron tran-
scripts, in contrast to reptilian, avian and mammalian
type I IFNs, which are encoded by intronless tran-
scripts.66,67 The type I IFNs in lower vertebrates could
not be phylogenetically classified as a or b, but a detailed
structural analysis in teleost fish has subdivided it into
groups I and II, based on the amount and position of
cysteine residues.67 The role of type I IFNs in antiviral
immunity has been corroborated in different vertebrates.
In teleost fish like the crucian carp (Carassius auratus L.)
these cytokines provide strong antiviral protection against
grass carp haemorrhagic virus infection and mediate the
Poly I:C-induced antiviral response, acting through the
signal transducer and activator of transcription 1 path-
way, as occurs in mammals.68 Likewise, type I IFNs have
been identified as repressors of viral protein synthesis and
stimulators of NK cells in amphibians69 and have been
recently evaluated in frog virus 3 (FV3)-infected frogs
(Xenopus laevis), demonstrating its capacity to inhibit the
replication of FV3 and expand the mean survival time of
infected tadpoles.66 In reptilians, IFN-like activity have
been reported in virus-infected animals and cell lines,3
but a detailed evaluation of type I IFNs in these verte-
brates is still missing. On the other hand, this type of
IFN has been characterized in birds, at genetic and
molecular levels (reviewed in ref 70). The presence of an
IFN-a homologue and other closely related genes was
reported in chickens, where they are encoded by a single
intronless transcript, like in mammals.70 These IFN-like
molecules showed significant antiviral properties in virus-
infected chicken embryos and cell lines and were also
identified in ducks and turkeys.25,70 A second type I IFN,
homologue to mammalian IFN-b and encoded by an
independent intronless transcript, was further identified
in chickens demonstrating antiviral activity in cell
cultures.71,72
Type II IFNs are also conserved among vertebrates and
their main functions include antiviral defence, regulation
of MHC expression, phagocytosis stimulation, inhibition
of cell growth and apoptosis.63–65,70 Interferon-c is the
only type II IFN identified in mammals, whereas in lower
vertebrates like teleost fish multiple type II IFNs have
been found.71 In Takifugu rubripes and other teleosts,
IFN-c homologues have been identified and they share
the antiviral properties and genomic organization of
mammalian IFN-c.72,73 Likewise, in the amphibian Xeno-
pus tropicalis the expression of the IFN-c gene has been
corroborated.74 This result was confirmed by Guanchun
and Jacques in Xenopus laevis, demonstrating that the
IFN-c gene is up-regulated by FV3 infection in tadpoles
and adult frogs.75 New evidence demonstrates the pres-
ence of an IFN-c gene in the North American green anole
132
lizard (Anolis carolinensis) and its expression in different
tissues.65 On the other hand, in different birds such as
chickens, ducks, turkeys, pheasants, quails and Guinea
fowls the presence of IFN-c genes with high sequence
homology to mammalian IFN-c and strong antiviral
activity as well as macrophage-stimulating capacity has
been reported.70,76 An increased expression of IFN-c gene
has also been detected in chickens during coccidial infec-
tions.25 In contrast, type III IFNs (IFN-k) have only been
identified in mammals and recently in the amphibian
Xenopus laevis.71 In both species IFN-k seems to be
involved in antiviral defence and displays common func-
tions with type I IFNs.65,71 Although type III IFNs are
not present in teleost fish,71 the existence of IFN-k genes
in reptilians or birds has not been properly evaluated.
Hence, a genomic screening for IFN-k in amniotes is cru-
cial to determine the evolution of type III IFNs in jawed
vertebrates.
Interleukins are another class of cytokines conserved in
vertebrate immunity, but they are not equally represented
in all classes of vertebrates. Interleukin-1, IL-2 and IL-8
are among the most preserved ILs in amphibians, reptiles,
birds and mammals.3,61,63,64,70,77 Additionally, IL-1 has
been identified in teleost fish with the same functions that
it exhibits in other vertebrate species.8,49 The mammalian
IL-1 cytokine family has three characterized members: IL-
1a and IL-1b, which mediate inflammatory responses and
leucocyte stimulation and a third member, the IL-1
receptor antagonist (IL-1Ra) that inhibits the biological
activity of IL-1a and IL-1b.1,64 Of these three genes, only
the IL-1b has been identified in other vertebrates. This
gene has been detected in 13 teleost species,8,50 in
amphibians75 and in chickens,25,70 were it preserves the
pro-inflammatory and cell-stimulating capacities that
have been observed in mammals. In reptiles an IL-1-like
molecule was detected but it could not be classified as a
or b.78 This cytokine is produced by splenic macrophages
of the wall lizard (Hemidactylus flaviviridis) in response
to lipopolysaccharide stimulation.78 On the other hand,
IL-2 that is involved in lymphoid and myeloid cell differ-
entiation in mammals63 has been identified in reptilians
like diadem snake (Spalerosophis diadema) as a thymocyte
proliferating factor3 and in chickens associated to the
nuclear factor-jB to regulate cell differentiation.70 Fur-
thermore, the IL-2 gene is over-expressed in amphibians
like Xenopus tropicalis, during Batrachochytrium
dendrobatidis infection.61
Interleukin-8 is another cytokine conserved in verte-
brate immunity. This molecule acts as a chemokine that
participates in macrophage and leucocyte chemoattrac-
tion.1,70,75,79 It has been identified in elasmobranchs like
dogfish (Triakis scyllia)80 and teleosts like Onchorhyncus
mykiss,81 reptiles like Pelodiscus sinensis,77 domestic
birds71 and mammals.1,79,80 However, no IL-8 homologue
gene has been reported in amphibians. Interleukin-6 is
ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139

another important cytokine in the immune response that
stimulates the proliferation of B lymphocytes and the
production of fibronectin and APPs in hepatocytes.64,82 It
has been found in fish,8,50 amphibians like Rana escu-
lenta,80 birds and mammals,70,79,80,82 but not in reptiles.
Other ILs such as IL-15, IL-16, IL-17 and IL-18 seem to
be restricted to birds and mammals and their functions
include cell differentiation, chemoattraction of T lympho-
cytes, induction of cytokine expression and stimulation of
IFN-c secretion.25,70,79,80,83,84 Additional ILs that display a
variety of functions in innate and adaptive immunity
have been identified in mammals. For example, IL-4, IL-
5, IL-10 and IL-13, which are secreted during T helper
type 2 response and regulate lymphoid cell activation and
cytokine secretion.64,79,85 Likewise, IL-12, IL-20,64 IL-21,86
IL-22, IL-26,73,87 IL-25 and IL-3385 have been character-
ized in mice or human tissues, where they exhibit specific
biological properties related with inflammation, leucocyte
proliferation and lymphocyte differentiation. Interestingly,
IL-12, IL-22 and IL-26 were also discovered in teleost
fish,73,80 suggesting that some of these cytokines could be
present in other vertebrates but remain unidentified.
Among pro-inflammatory cytokines, TNF-a is the sec-
ond most conserved across vertebrate species after IL-1b
and is implicated in leucocyte chemoattraction and
macrophage stimulation.8,25,63,64,79 This cytokine has been
detected in teleosts such as rainbow trout (Onchorhynchus
mykiss), sea bream (Sparus aurata), goldfish (Carassius
auratus) and catfish (Ictalurus punctatus),8,49 in amphib-
ians like Xenopus laevis75 and mammals.63,64,79 A recent
work identified the TNFSF13 gene (a member of the TNF
superfamily) in Xenopus laevis and demonstrated that its
encoding protein promotes the proliferation of Xenopus B
lymphocytes and mouse splenic B cells.88 The similar
structure, tissue expression and bioactivity of TNFSF13
between mammals and Xenopus laevis, suggest that this
anuran could be an interesting model to evaluate different
immunological disorders caused by this cytokine.88 On
the other hand, although TNF-a-like activity was reported
in chickens, the sequence of a chicken TNF-a homologue
gene has not been identified or described in any data-
base.25,70 Similarly, the presence of this molecule or its
encoding gene has not been reported in reptiles.
Other immunoregulatory cytokines like TGF-b80 are
also well-preserved across vertebrate species and are pre-
sent in early metazoans and protozoans.89 The mam-
malian TGF-b family have been extensively characterized
and include three related isoforms encoded by indepen-
dent genes, that regulate cell growth and differentiation
and are involved in oocyte maturation, wound repair,
modulation of inflammatory response, immunosuppres-
sion and tolerance.90 In the same way, the presence of
TGF-b in other vertebrates has been reported and the
ubiquitous expression of this pleiotropic cytokine among
animals could be more related to its function in repro-
ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139
Innate immunity in vertebrates
duction and embryonic development than to its role in
immunity.80 In 2009, a genomic screening of 33 animal
species of different taxa identified the TGF-b gene in
humans, primates such as Pan troglodytes and Macaca
mulatta, rodents like Mus musculus and Rattus norvegicus,
in dogs (Canis domesticus), birds like roosters (Gallus gal-
lus), osteichthyes (bony fish) such as Oryzias latipes,
Danio rerio, Tetraodon nigroviridis and Takifugu
rubripes.89 The TGF-b is also secreted by the placenta of
viviparous squamate reptiles like Chalcides chalcides.80
Additionally, the three mammalian isoforms of TGF-b
were identified in chickens as regulators of embryonic
development and haematopoiesis.70 Interestingly, the
mRNA expression of a fourth isoform, TGF-b4, is
induced by coccidial infection in the intestinal epithelia
of chickens.25,70
Natural antibodies
In the plasma of vertebrates there are circling antibodies
known as natural antibodies (NAbs) (Fig. 1), which are
mainly of IgM isotype, possess a limited genetic repertoire
and are synthesized by B cells in absence of pathogens.91
These pre-existent immunoglobulins are essential compo-
nents of humoral innate immunity; they react against for-
eign antigens and microbe-derived substances and can
activate the complement cascade by the classical path-
way.60,91
The NAbs have been described as part of humoral
immunity in fish, reacting with proteins exposed on the
microbial surface and providing protection against patho-
gens like Aeromonas salmonicida.92 This natural IgM is
found in chondrichthyes (sturgeons and sharks) and some
osteichthyes such as Onchorhynchus mykiss and Pagrus
auratus.93 Such IgM isotype NAbs have also been identi-
fied in amphibians,94 where they are implicated in the
inactivation of modified bacteriophages and other viral
particles,95 as well as haemoagglutination of foreign red
blood cells from chicken, sheep, rat and human.96 High
levels of NAbs have been detected in anurans like Bufo
regularis, Bufo arenarum and Xenopus laevis.96
The production of NAbs is also present in reptiles, as
an essential element of the humoral immune response
that has been found in some species, e.g. water python
(Liasis fuscus).97 This study demonstrates that NAbs
increase with age for tropical pythons, possibly to com-
pensate for the immunosenescence and loss of effective-
ness in the adaptive humoral immune response.97 Natural
immunoglobulins like IgM and IgG, are also produced by
the gut-associated lymphoid tissue of birds, due to stimu-
lation by the intestinal microbiota.98 These NAbs elimi-
nate bacteria and spirochaetes in vivo and promote the
clearance of bacterial substances like lipopolysaccha-
rides.60 They represent an important humoral defence
mechanism that correlates with resistance to pathogens,
133
M. Riera Romo et al.
like Amblyceran lice in some bird species such as
Gal
apagos hawks (Buteo galapagoensis).99 In addition,
NAbs against parasites like Plasmodium lophurae have
been identified in White Leghorn chickens (Gallus domes-
ticus).60
The NAbs are present in mammals as well, representing
a conserved element from lower vertebrates involved in
humoral innate immunity; they neutralize pathogens and
helminth parasites2 and confer resistance against influenza
virus.91 These antibodies also display new modulatory
functions in complex vertebrates like mammals. For
example, the binding to oxidized proteins like oxidized
low-density lipoprotein, in a mechanism that reduces its
uptake by macrophages and attenuates the atherogenic
process.100 Additionally, some reports demonstrate that
these natural IgMs participate in immune surveillance,
recognizing tumour-associated or tumour-specific anti-
gens such as CFR-1, expressed in malignant cells of differ-
ent epithelial cancers, and even inducing apoptosis.101
Cell-mediated innate immunity
Intraepithelial T lymphocytes
The mucosa and epithelial barriers of vertebrates are infil-
trated by a specific type of lymphocyte – intraepithelial T
lymphocytes (ITLs) (Fig. 1), which recognize and destroy
pathogens.102 These cells have been better characterized in
mammals and are phenotypically classified as cd T lym-
phocytes.11,102 However, they have more characteristics in
common with the effector cells of innate immunity. For
example, its limited antigenic repertoire and the low vari-
ability of its T-cell receptors make them different from
the typical lymphocytes present in the adaptive immune
response.11,102
Intraepithelial lymphocytes have been isolated from the
intestinal epithelial tissue of fish, like Onchorhynchus
mykiss, and its cytotoxic capacity against EL4 mouse thy-
moma cells has been demonstrated.27 Likewise, in some
specialized epithelia of amphibians, such as the gut-asso-
ciated lymphoid tissue, the presence of intraepithelial
lymphocytes has been reported; these are similar to those
found in fish and share many phenotypic characteristics
with the ITLs of mammals.26
These lymphocytes have also been identified in reptiles,
as an element of protective immunity, in intestinal epithe-
lium and gut-associated lymphoid tissue,28 a characteristic
that is preserved from fish and amphibians. They are pre-
sent in some species, such as platypus (Ornithorhynchus
anatinus), in the lamina propria of intestinal mucosa, and
are phenotypically similar to mammalian ITLs.28 The gut-
associated lymphoid tissue of birds is also infiltrated by
intraepithelial lymphocytes, which diversify into various
subsets of T lymphocytes that include CD8+ T cells
(CD8aa), and cd T CD8ab cells.29 These cells represent a
134
primary active barrier against pathogen dissemination, and
constitute the main component of protective immunity to
avian coccidiosis caused by the intestinal parasitic patho-
gen Eimeria.25 Some investigations indicate that this cellu-
lar component of epithelium increases in number after
infection with intestinal parasites such as Eimeria acervu-
lina and Eimeria tenella.103
In humans, two subsets of cd T lymphocytes have been
identified: Vd1 and Vd2 cd T cells. Vd1 T cells reside
mainly in the epithelial mucosa, whereas Vd2 T cells are
abundant in the lymphatic system and peripheral blood.11
These cells can recognize non-peptidic antigens, soluble
proteins and MHC molecules, displaying multiple func-
tions, such as cytokine secretion, activation of dendritic
cell maturation, macrophage recruitment, cytotoxic activ-
ity against transformed cells, maintenance of epidermal
integrity and B- or T-cell cooperation, among others.11
Myeloid phagocytic cells
The main cellular components of vertebrate innate immu-
nity are myeloid phagocytic cells, also called ‘professional’
phagocytes3,94 (Fig. 1). This type of leucocyte includes
monocyte–macrophages, neutrophils/heterophils, baso-
phils and eosinophils.1–3,94 These cells phagocytose and
destroy pathogens, secrete cytokines and release soluble
mediators like histamine, reactive oxygen species, reactive
nitrogen species, lysozyme and AMPs.2,3,79,104 They recog-
nize senescent cells and microbes by pattern recognizing
receptors and present phagocytosed antigens to B or T
cells, acting as the first line of defence against infection
and contributing with the activation of adaptive immune
response.1,79,104 The main pattern recognizing receptors
expressed in vertebrate phagocytes are the Toll-like recep-
tors, C-type lectin receptors, NOD-like receptors and Rig
I-helicases, which recognize chemical structures that are
present in all microbes, such as lipopolysaccharides,
lipoproteins, complex glycans, formylmethionine and
even nucleotides and nucleic acids.5–7,12 These receptors
are able to distinguish between chemical isomers and can
also recognize a variety of polysaccharides, lipids or
nucleotides, cooperating with each other to selectively
activate different signalling pathways that induce specific
pro-inflammatory and antimicrobial innate responses.5,6
Myeloid phagocytes represent a potent defence
mechanism in mammals, essential against infections with
Gram-negative (Escherichia coli, Salmonella typhimurium,
Yersinia enterocolitica) and Gram-positive (Listeria
monocytogenes, Staphylococcus aureus) bacteria.104,105 The
immune functions of mammalian innate myeloid cells
have been evaluated in mouse models and they include
phagocytosis, antigen presentation, releasing of histamine,
pro-inflammatory cytokines, AMPs and granule-derived
proteins that destroy microorganisms.1,104,105 These cells
are also involved in wound healing and normal tissue
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functions.106 Among myeloid phagocytic cells, neutrophils
represent the earliest innate response in humans, phago-
cytosing microbes, cooperating with other immune cells
and releasing AMPs, proteases, hydrolytic enzymes and
phospholipases.105,107
In other vertebrates, leucocytes such as macrophages,
heterophils (homologues of mammalian neutrophils),
basophils and eosinophils are crucial for the effectiveness of
innate immunity.3,94,108 These cells have been described in
fish,8,108,109 amphibians23,75 and reptiles,3,78,110 preserving
their functions in phagocytosis, inflammation and the
release of soluble mediators. In these ectothermic verte-
brates, the phagocytosis plays an important role in immune
response, because it is less influenced by temperature than
adaptive immune mechanisms.111 Recent imaging tech-
niques in fish model organisms like Danio rerio, reveal new
insights into the phagocytotic process and immune cell
recruitment during inflammation, from a unique in vivo
perspective.108 These approaches combined with genetic
manipulation have allowed the discovery of novel inflam-
matory pathways, danger signals and myeloid develop-
ment-related genes, which are integrative findings for the
comprehension of the mammalian immune system.108
Innate myeloid cells have also been characterized in
birds. They include macrophages, heterophils, eosinophils
and basophils, which are preserved from the early verte-
brates and whose main function is the recognition and
phagocytosis of pathogens.112 Its pattern recognizing
receptor-mediated recognition stimulates phagocytosis
and activates destruction of pathogens, antigen presenta-
tion, stimulation of the adaptive immune response and
production of pro-inflammatory cytokines.112
On the other hand, the lymphoid lineage of vertebrate
leucocytes has recently been a focus of research in innate
immunity to comprehend the developmental relationships
between the various lineages that have been identified to
date as well as their functionality. A new type of leuco-
cyte, known as a nuocyte, has been identified in mam-
mals, implicated in type 2 immunity against helminth
parasites.85 These cells were characterized in the IL13-
eGFP reporter mice, demonstrating an IL-25/IL-33-depen-
dent proliferation and a capacity to secrete IL-13 during
infection with the helminth Nippostrongylus brasiliensis.85
In addition, recent reports describe the existence of lin-
eage-marker-negative innate lymphoid cell subsets that do
not express cell surface markers associated with other
immune cell lineages. These newly identified members of
the lymphoid lineage, which include nuocytes, have
emerging roles in mediating immune responses and in
regulating tissue homeostasis and inflammation.113
Phagocytic B cells
The B lymphocytes are considered another type of cell
involved in vertebrate innate immunity (Fig. 1). In lower
ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139
Innate immunity in vertebrates
vertebrates these cells display phagocytic capacity and
activation of degradative pathways that result in
phagolysosome fusion and killing of internalized bacte-
ria.94,114 B cells seem to act like leucocytes; however, they
are able to produce antibodies of IgG and IgA isotype,
involved in classical adaptive immune response, and also
NAbs of IgM isotype.92,110
Phagocytic B cells have been identified in teleost fish
like Onchorhynchus mykiss and Ictalurus punctatus, in
amphibians like Xenopus laevis and in reptiles like the
red-eared slider (Trachemys scripta).110,114 In mammals,
the B cells do not exhibit phagocytic capacity and their
immune functions seem to be limited to adaptive immu-
nity.94,110,114 However, a mammalian subpopulation of B
lymphocytes known as B-1 lymphocytes, retains the
phagocytic capacity and antimicrobial properties of the B
cells from lower vertebrates.114 These cells reside mainly
in the peritoneal and pleural cavities91 where they pro-
duce NAbs and present phagocytosed antigens to CD4+ T
cells, playing an important role in innate and adaptive
immune responses.114 The phagocytic ability of non-
mammalian B cells which is preserved in B-1 cells, sug-
gests an evolutionary connection between both cell types.
In fact, the discovery of a bipotent B macrophage precur-
sor in fetal liver and adult bone marrow of mammals,
supports the idea of a common origin for B cells and
macrophages, from a phagocytic ancestor.94,114 In con-
trast, the phagocytic capacity of B cells in birds has not
been evaluated, making it hard to determine the evolu-
tion of this function into the higher amniotes.110 How-
ever, subsets of B cells, similar to B-1 lymphocytes of
mammals, are found in birds, in the gut-associated lym-
phoid tissue where they produce NAbs.98
Non-specific cytotoxic cells
Non-specific cytotoxicity is one of the most important
cellular mechanisms in vertebrate innate immunity
(Fig. 1), playing an essential role in antiviral defence and
immune surveillance.115,116 This function is carried out in
mammals mainly by NK lymphocytes, which are involved
in the cytotoxic response against cells that are trans-
formed or infected by virus or intracellular microbes.115
In addition, NK cells secrete cytokines with antiviral
activity117 and are also involved in hepatic defence and
tissue regeneration and remodelling.87
The presence of non-specific cytotoxic cells similar to
NK cells has been described in lower vertebrates. These
cells have been identified in fish like Ictalurus punctatus
where they are distinguishable by their small, non-granu-
lar morphology and their abundance in lymphoid tissues,
such as kidney and spleen.8,118 In addition, the identifica-
tion of a new gene family known as novel immune-type
receptors in zebrafish (Danio rerio) is strong evidence for
the presence of NK-like cells in teleosts.108 These proteins
135
M. Riera Romo et al.
are structurally similar to mammalian NK receptors, are
differentially expressed in fish lymphoid cells but not in
myeloid cells, and confer lytic capacity on the cells in
which they are expressed.108
Natural killer-like lymphocytes have also been reported
in amphibians, with an essential role in cytotoxic
response against virus-infected cells.69,75 Many NK candi-
dates have been identified in the intraepithelial layer and
the spleen tissue of Xenopus laevis, using a staining
method with specific anti-NK monoclonal antibodies 1F8,
1G5 and 4D4.119 This study demonstrates the existence of
at least three subsets of NK lymphocytes in Xenopus lae-
vis: 1F8+ CD5– splenocytes, 1F8+ CD5lo gut cells and
1F8+ F17– lymphocytes, which exhibit strong in vitro
cytotoxic activity, against the Xenopus thymus lymphoid
tumour cell line B3B7.119 These results have been con-
firmed by Goyos and Robert in 2009,116 through the
identification of 1F8+ NK cells and NKT-like cells (with
NK- and T-cell-associated markers), which are similar to
those found in mammals and are involved in in vivo anti-
tumour immune responses in Xenopus laevis. Different
studies in this anuran species have revealed the connec-
tion between tumorigenesis and embryonic development
and are also providing evidence for the innate cellular
responses to oncogenic processes.116 In comparison to
murine models, anurans like Xenopus laevis and Xenopus
tropicalis provide advantages in the genetic manipulation
and constitute a powerful alternative for the identification
of new genes involved in tumorigenesis and anti-tumour
immune responses.116
In birds, non-specific cytotoxic activity against infected
or transformed cells has also been reported, carried out
by NK-like lymphocytes called TRC0 cells.120 These cells
are homologues to mammalian NK lymphocytes and
express their typical surface antigens, such as CD3.120
This kind of lymphocyte has been identified in the
intestinal mucosa of chickens, where they are subdivided
into two distinct populations: 28-4+ CD3– NK cells (de-
tected with 28-4 monoclonal antibody) and 28-4+ CD3+
NK cells, which are homologues to the NKT cells of
mammals.121 They represent a conserved cellular element
from the lower vertebrates that plays an important role in
anti-parasitic immune response and mucosal immunity.25
In contrast with other vertebrates, non-specific cyto-
toxic cells or NK cells are not found in reptiles, or at least
have not been identified and studied thoroughly. They
are present in the other classes of vertebrates, suggesting
that they should exist in reptiles as well. This theory can
be supported by the finding of NK-mediated cytotoxicity
against tumour cells in the turtle Mauremys caspica.122
This activity was identified in thymic cells, but was not
defined in a specific subset.122
In addition, a particular type of NK cell, with many
phenotypic characteristics of a T lymphocyte, known as
NKT lymphocyte, has been described in mammals.117,123
136
These cells recognize glycolipid antigens in the context of
the CD1 molecule and release cytokines implicated in the
innate immune response.123 The presence of NKT func-
tional analogues has been reported in amphibians116 and
birds,25,121 suggesting that this subset of NK lymphocytes
is a conserved component among vertebrates, which rep-
resents an evolutionary link between innate and adaptive
immunity. The main functions of these cells include cyto-
toxic response, antitumour activity123 and the secretion of
antiviral cytokines like type I IFNs117 and IL-21.86 Other
types of invariant T cell, similar to NKT lymphocytes, are
known as mucosal-associated invariant T cells and are
also implicated in the mammalian innate immune
response by the release of IL-17, mainly in the gastroin-
testinal tract84 and the liver.124
Recent comparative studies across animal species have
revealed new functional properties for NK lymphocytes
and other innate cells, like monocyte–macrophages.13,14
These novel findings indicate that innate immune cells
display enhanced responses to secondary infections in a
T/B-cell-independent manner, through epigenetic mecha-
nisms and a biochemical reprogramming that predisposes
cells to a rapid activation by secondary stimulation.13,14
This innate immunological memory termed as ‘trained
immunity’ is an interesting new feature of innate cells
that is changing the concept of innate immunity.13,14
Conclusions
Across jawed vertebrates, innate immunity displays a
diversity of mechanisms that adapt to the anatomy and
behaviour of different species, while preserving its core
elements and main functions. Physical barriers are con-
served across all vertebrates, nevertheless they have
anatomical and chemical variations that exhibit a ten-
dency to specialization of the skin and mucosa. Besides,
among humoral components lysozyme, AMPs, comple-
ment system and NAbs are the most conserved, showing
the same basic functions and sharing their main proper-
ties. On the other hand, the acute phase response and
cytokine release are conserved mechanisms, triggered in
the presence of pathogens, even though the involved
molecules differ among vertebrate species. In general,
humoral components are poorly characterized in amphib-
ians and reptiles, making it difficult to understand the
evolution of humoral innate immunity in vertebrates.
The current evidence reveals non-mammalian verte-
brates as a source of potent antimicrobial and antiviral
proteins and peptides, which could also have antitumour
or immunomodulatory activity. There are several exam-
ples of antimicrobial molecules isolated from inverte-
brates and plants with potential applications in
biomedicine; however, vertebrates are an interesting alter-
native for the development of new therapeutic agents to
deal with antibiotic resistance and chemotherapy toxicity.
ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139

Cell-mediated components of innate immunity are pre-
served across vertebrates and they include ITLs, myeloid
phagocytes and non-specific cytotoxic cells. Nevertheless,
the existence of reptilian non-specific cytotoxicity remains
unclear. Further investigation and characterization of cell-
mediated immunity in reptiles is required to elucidate the
transition of non-specific cytotoxic cells into the
amniotes. On the other hand, the B lymphocytes are also
described as innate effector cells, a recent concept in ver-
tebrate immunity. B cells produce NAbs and display
phagocytic capacity and antimicrobial properties in lower
vertebrates, a characteristic that remains in mammalian
B-1 lymphocytes. These findings suggest an evolutionary
connection between macrophages and B cells, which
could have evolved from a common phagocytic ancestor.
However, the phagocytic capacity of B cells from birds
has not been examined. A detailed evaluation of phago-
cytic and antimicrobial characteristics of avian B cells is
the key to trace the evolution of B-cell-mediated phago-
cytosis into the higher amniotes.
In spite of the evident physiological and morphological
differences among the five classes of jawed vertebrates,
that determine the kinetics and overall effectiveness of
immune responses, the molecules and cells involved in
the innate response are conserved and maintain their
functionality throughout vertebrates. Hence, even if the
immunity in higher vertebrates is more complex than in
lower vertebrates, they share some of the basic mecha-
nisms involved in cutaneous protection, serum antimicro-
bial activity, acute phase response, opsonization,
pathogen recognition, phagocytosis and cytotoxicity,
among others. From this point of view, non-mammalian
vertebrates can be considered as models for the study of
the immune system. In this sense, different zebrafish
mutants have been used to examine key aspects of
immune system ontogeny, phagocytosis, cell recruitment
and inflammatory pathways, while the frog Xenopus laevis
is a potent model of tumorigenesis, tumour immunity
and other immune-related pathophysiological conditions.
Likewise, chickens and other domestic birds constitute
models for the study of the gut-associated lymphoid tis-
sue and the pathogen–host interactions at the intestinal
epithelium.
Further investigation is required to achieve the com-
plete characterization of innate immune mechanisms and
their evolution across vertebrates. This could lead to a
better understanding of the cellular and molecular pro-
cesses that determine the dynamic functionality of innate
immunity.
Disclosures
The authors declare that there are no financial or com-
mercial conflicts of interest associated with this work.
ª 2016 John Wiley & Sons Ltd, Immunology, 148, 125–139
Innate immunity in vertebrates
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